Experimental Study Of Glutamate Inducing Proliferation Of Adult Neural Stem Cells Via Sonic Hedgehog Signaling Transduction Pathway

Background Treatment of cerebral ischemia is always a difficult problem in the field of medicine,traditional treatment can not repair the damaged brain tissue,the discovery of neural stem cells (neural stem cells, NSCs) provides a new idea to solve this complex problem, but the many complex mechanism in the neural stem cells of survival and other aspects make the application research in this field still not obtain breakthrough progress,the resting state neural stem cells are activated in situ and start to proliferate after ischemic cerebral infarction, glutamate (Glutamate, Glu) concentration of cerebral infarction area rises, and Shh (Sonic Hedgehog) protein expression also increases;considering that Shh signaling pathway plays an important role in the proliferation of embryonic stem cells,so we speculate that glutamate mediates the proliferation of neural stem cells via the Shh signaling pathway. This paper plan to explore preliminarily the relationship between Shh expression and Glu stimulated adult neural stem cells proliferation in vitro.The subject is divided into three parts as following.Part I Improvement of isolation,culture and identification of neural stem cells from adult SD ratObjective To improve method for isolating,culturing and identifying neural stem cells derived from the hippocampus dentate gyrus in adult SD rat.Observe characteristics of growth,multiplication and differentiation.and prepare neural stem cells for subsequent experiments.Methods To separate the whole hippocampus from adult SD rat.make use of mechanical blow to acquire primary cell.disperse the neural cell pellet when passage by accutase digestion method, proliferation of neural stem cells can be determined by cck-8 method.Cultured and differentiated cells were identified with multiple immunofluorescence cytochemistry method.Results Primary neural stem cells can be obtained by mechanical blow,accutase digestive method is more conducive to the neural stem cells in the process of subculture,the CCK-8method is simple to determine the proliferation of neural stem cells,multiple immunofluorescence dynamically display the differentiation process of neural stem cells after being induced.Conclusions The improved method is more simple and efficient in obtaining and culturing a large number of cells,The isolated and cultured cells are determined to are neural stem cells by multiple immunofluorescence.Part Ⅱ Experimental study of the optimum concentration of glutamate induced proliferation of adult neural stem cellsObjective inducing growth of adult neural stem cells by various concentrations of glutamate find out the optimum concentration of glutamate that stimulated neural stem cells proliferation,and preparing for the subsequent experiments.Methods More than3passages of neural stem cells were cultured for24h,48h,72h in neural stem cell culture medium that containing different concentrations of glutamate and removing growth factor,and the neural stem cells proliferation of different glutamate concentrations were detected by CCK-8method.Results Glutamate of the range of0μmol/L～100μmol/L concentration can induce proliferation of adult neural stem cells,20μmol/L glutamate concentration is most suitable for the proliferation of adult neural stem cells, more than100μmol/L glutamate concentration can inhibit the proliferation of adult neural stem cells, and have certain cytotoxicity on adult neural stem cells.Conclusions A range of concentrations of glutamate (0μmol/L-100μmol/L) can stimulate adult neural stem cells growth in vitro culture conditions,20μmol/L glutamate concentration is most suitable for the proliferation of adult neural stem cells, more than 100μmol/L glutamate concentration will inhibit the proliferation of adult neural stem cells, and have certain cytotoxicity on adult neural stem cells.Part Ⅲ Glutamate promote adult neural stem cells proliferation through Sonic Hedgehog signaling pathwayObjective Inducing proliferation of adult neural stem cells with20μmol/L glutamate explore the relationship between Shh expression and glutamate stimulating adult neural stem cells proliferation.Methods More than3passages of neural stem cells were cultured for24h,48h,72h in final concentration of20μmol/L glutamate neural stem cell culture medium without growth factor,set to glutamate stimulation of24h,48h,72h3groups.and were cultured for72h in no glutamate neural stem cell medium without growth factor,as the blank control group.According to the method of grouping sets above,set the same group and respectively add fetal bovine serum to induce different for lweek.observe growth of neural stem cells and induced differentiation cells.Expression of nestin protein,Shh protein,GFAP protein and βⅢ-tubulin protein were detected by western-blot method.make use of real-time fluorescent quantitative method to detect the expression of nestin mRNA,Shh mRNA,GFAP mRNA and βⅢ-tubulin mRNA.Results In the process of the glutamate stimulating proliferation of adult neural stem cells,nestin expression and Shh expression changes consistent,neural stem cells differentiate into neurons and glial cells after being induced differentiation for lweek.Conclusions Shh signal pathway is involved in the process of glutamate inducing neural stem cells growth,Glutamate does not affect the differentiation potential of adult neural stem cells.